Rapid, reliable and inexpensive characterization of polymers, particularly nucleic acids, has become increasingly important. A high-throughput device that can probe and directly read, at the single-molecule level, hybridization state, base stacking, and sequence of a cell""s key biopolymers such as DNA, RNA and even proteins, will dramatically alter the pace of biological development.
Church et al. in U.S. Pat. No. 5,795,782 recently reported that a voltage bias could drive single-stranded charged polynucleotides through a 1-2 nanometer transmembrane channel in a lipid bilayer. Data in the form of variations in channel ionic current provide insight into the characterization and structure of biopolymers at the molecular and atomic levels. The passage of an individual strand through the channel could be observed as a transient decrease in ionic current. Experiments using biological membranes and pores have demonstrated extraordinary electronic sensitivity to the structure of translocating molecules. See, U.S. Pat. No. 5,795,782 and Kasianowicz et al. (xe2x80x9cCharacterization of individual polynucleotide molecules using a membrane channelxe2x80x9d, Proc. Natl. Acad. Sci. 93:13770 (November 1996)).
This is demonstrated in FIG. 1, in which a lipid bilayer 10 having a xcex1-hemolsin channel 12 therein is shown. A Staphylococcus aureus xcex1-hemolsin channel is used because its inner diameter has a limiting aperture of 1.5 nm, which is adequate to admit single-stranded DNA. The layer separates two solution-filled compartments 14, 16 in which ions are free to migrate through the channel 12 in response to an applied voltage. The unobstructed ionic current 18 is illustrated in the upper channel 12 of FIG. 1. If negatively charged molecules, such as DNA, are placed in compartment 14 and a negative bias is applied, the molecules are pulled one at a time into, and through, the channel. The ionic current is reduced as a polymeric molecule 17 traverses the channel from the cis to the trans compartment, as is illustrated in the lower channel 19 of the figure. The number of transient decreases of ionic current per unit time (the blockade rate) is proportional to the concentration of polymer in the source solution. Furthermore, the duration of each blockade is proportional to polymer length.
FIG. 2 is an example of actual current traces obtained using a lipid membrane containing an S. aureus xcex1-hemolsin channel. The voltage applied across the bilayer (xe2x88x92120 mV) produces a current of ions that flow through the channel. After adding DNA, transient reductions in current are evident in the trace (FIG. 2A). The time it takes for the DNA to be drawn through the channel (FIG. 2B), effectively measures the length of a DNA molecule (here, 1300 xcexcs corresponding to a 1,060 nt polymer). The extent to which ionic flow is reduced (here, from about 120 xcfx81A to 15 xcfx81A) reflects the physical properties of the nucleotides in the polymer.
While a protein channel has demonstrated the ability to identify characteristics of polynucleotides, attaining the resolution and precision needed to achieve error-free sequencing of individual monomers has proved to be a challenge. For example, it has been demonstrated that detection sensitivity extends along the entire length of the xcex1-hemolsin protein channel, and this despite the sharp limiting asperity of 1.5 nm at its neck. The interactions of multiple monomer units along its entire length contribute to the blocked current magnitude, thereby making it difficult to obtain unambiguous resolution of individual monomers characteristics.
The currently available biological pore membrane system suffers from a number of additional disadvantages, including limited temperature and bias voltage operating ranges, limited chemical environment accommodation, limited device lifetime due to pore diffusion in the membrane, high electronic noise levels associated with large membrane capacitance, and limited availability of pores with the desired diameter and lengths on the 1-10 nm scale. In order to maximize the capabilities of the present technology, certain advances in the technology are required.
The present invention provides methods and apparatuses based upon solid-state materials for molecular detection. In addition to providing remedies for the above problems associated with biological pores, a solid-state system for molecular detection offers the ability to provide and accommodate local, embedded conducting electrodes and xe2x80x9con chipxe2x80x9d integrated electronics that can extend the capabilities of xe2x80x9cionicxe2x80x9d current measurements and also offer the prospect of local and very sensitive electronic sensing by mechanisms such as injection tunneling spectroscopy.
In general, the method and apparatus of the invention provide for the traverse of individual monomers of DNA or any other linear polymer molecule across or through a limited volume in space in sequential order, preferably on the nanoscale range, e.g., a volume on a scale which accommodates a single monomer for interacting with a detector such as 1-10,000 nm3, and preferably 1-1000 nm3. The limited space reduces background noise associated with polymer detection, so that subtle differences in structure may be observed. The use of a limited volume also ensures that the monomers move in single file order.
In one aspect of the invention, evaluation of a polymer molecule including linearly connected monomer residues is accomplished by contacting a polymer-containing liquid with an insulating solid-state substrate having a detector capable of detecting polymer molecule characteristics, and causing the polymer molecule to traverse a limited volume on the solid-state substrate so that monomers of the polymer molecule traverse the limited volume in sequential order, whereby the polymer molecule interacts linearly with the detector and data suitable to determine polymer molecule characteristics are obtained.
In another aspect of the invention, evaluation of a polymer molecule including linearly connected monomer residues is accomplished by contacting a polymer-containing liquid with an insulating solid-state membrane having an aperture therein, wherein the aperture includes an entry port and an exit port defining a channel there between, and causing the candidate polymer molecule to traverse the aperture of the membrane, whereby the polymer molecule interacts linearly with the aperture and data suitable to determine polymer molecule characteristics are obtained.
xe2x80x9cSolid-statexe2x80x9d is used herein to refer to materials that are not of biological origin. By biological origin is meant derived from or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure. Solid-state encompasses both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si3N4, Al2O3, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon(copyright) or elastomers such as two-component addition-cure silicone rubber, and glasses, although there is no specific limitation to the materials that may be used according to the invention.
A xe2x80x9csolid-state substratexe2x80x9d of the invention is an insulating material, which is integratable with the electronic devices, e.g., electrodes, necessary to monitor and detect polymer interactions at the solid-state substrate surface. A solid-state substrate is not required to have an aperture.
A xe2x80x9cmembranexe2x80x9d is a layer prepared from solid-state materials, in which one or more apertures is formed. The membrane may be a layer, such as a coating or film on a supporting substrate, or it may be a free-standing element. Alternatively, it may be a composite of various materials in a sandwich configuration. The thickness of the membrane may vary, and in particular, the membrane may be considerably thinner in the region containing the aperture. In embodiments, in which the membrane is a layer on a supporting substrate, the supporting substrate includes an appropriately positioned gap, so that the portion of the membrane containing the aperture spans the gap.
An xe2x80x9caperturexe2x80x9d of the invention is an opening in a membrane that forms a pore, hole, or channel and is defined by its diameter, length and internal contour. The geometry is not crucial, except for that some constricting asperity may be provided somewhere either at the [rim] periphery or at a point through its length in some embodiments. The walls of the aperture should be electrically insulating; however, it is not required that the entire membrane containing the aperture be insulating, i.e., the membrane in which the aperture exists could be insulating, or the membrane may be conducting, and the aperture walls and membrane may be coated with an insulating material.
xe2x80x9cConstraining diameterxe2x80x9d is used herein to mean the smallest diameter of the aperture or the channel defined thereby, or an aperture-biomolecule composite. The constraining dimension may arise from an asperity or constriction region within the periphery of the channel. It may be defined by the entirety of the channel if the channel length is sufficiently short. It may be defined by a biomolecule; e.g., a material of xe2x80x9cbiological originxe2x80x9d as defined herein, which is adjacent to, above, below, or within the membrane aperture. By way of example, see FIGS. 13 and 14. For many embodiments, the length of the constraining diameter feature should be commensurate with the distance between individual monomers of the polymer molecule, e.g., the distance between nucleotides, so that only a single monomer at a time is capable of interacting within the constrained dimension of the aperture. Conducting electrodes may be provided on one or both sides of the membrane to enable detection of species through the aperture when electronic sensing is desired, or to apply a potential to the apparatus. The constraining dimension may also refer to the dimensions of the gap defined by opposing electrode tips or probes, when electrodes are used in an electronic sensing mode.
xe2x80x9cTime-dependentxe2x80x9d interaction is used herein to mean those types of interactions between the polymer molecule and the detector, e.g., the constraining dimension of the aperture or the electrode tip of the electrodes, and the like, which are time-dependent or monitored as a function of time. For example, the length of a polymer may be related to the time of a single current blockade event. Another time-dependent interaction may be the number of current blockade events per unit time, which is an indication of the number of polymer molecules in solution. Thus, polymer size and polymer concentration also may be considered time-dependent interactions.
xe2x80x9cMonomer-dependentxe2x80x9d interaction is used herein to mean those types of interactions between the polymer molecule and the detector, e.g., the constraining dimension of the aperture or the electrode tip of the electrodes and the like, which are determined by the nature of the monomer. For example, the chemical composition of individual monomer may be detected as each monomer passes by and interacts at the detector. Thus, polymer monomer identification, e.g., DNA sequencing, is a monomer-dependent interaction.
In preferred embodiments, the channel is coated with an electrically insulating layer or with a passivating layer. The solid-state may be selected from the group consisting of inorganic compounds, organic and inorganic polymers and glasses, and may selected from the group consisting of silicon nitrides, silica, alumina. In preferred embodiments, the solid-state membrane has a thickness in the range of about 10 nm to about 1 mm, and preferably in the range of about 50 nm to about 100 nm. In other embodiments, the solid-state membrane has a capacitance of less than about 0.1 xcfx81F.
In other preferred embodiments, the aperture includes a constraining diameter, and the constraining diameter is in the range of less than about 20 nm, preferably less than about 5 nm, and more preferably in the range of about 1-2 nm. The constraining diameter may include a feature integral with the aperture and tapering acutely from a point of constriction in the aperture channel and that taper may be curvilinear, or the taper varies in acuteness along the length of the channel. In some embodiments, the feature is located at the exit or entry port of the aperture, within the channel of the aperture. In other embodiments, the length of the constraining diameter is in the range of 1 to 10 nm, and preferably in the range of 1 to 5 nm.
In other embodiments, the detector comprises first and second electrodes adjacent to the aperture and in electrical communication with the channel. The first and second electrodes are on the same side of the solid-state membrane, or the first and second electrodes are on opposing sides of the solid-state membrane. The electrodes may be a conductive metal layer deposited on the solid-state membrane.
In other embodiments, the detector comprises the constraining diameter of the aperture. In still other embodiments, a polymer replicating catalyst is in contact with the aperture, and the polymer replicating catalyst is located adjacent to, above, below, or within the membrane aperture. The polymer replicating catalyst contains may include a constraining diameter feature.
In other embodiments, the monitoring means includes an ammeter or an electrometer.
In still other embodiments, the means for causing a candidate polymer molecule to traverse the aperture is selected from the group consisting of voltage gradient means and biomotors.
The apparatus may further include at least one insulating layer adjacent to the first and second electrodes, or a substrate supporting the solid-state membrane.
In another aspect of the invention, an apparatus for use in evaluating a linear polymer molecule is described having a first vessel having a first inlet therein, a second vessel having a second inlet therein, and an elongated cylinder having first and second ends, each end in sealing communication with the respective inlets of the first and second vessels. A solid-state membrane containing an electrically insulating aperture therein is disposed in the first end of the elongated cylinder, wherein the aperture includes an entry port and an exit port defining a channel there between, and the membrane is positioned to be contactable with a liquid containing a candidate polymer molecule in the first vessel. Means for causing a candidate polymer molecule to linearly traverse the aperture and a detector for detecting time-dependent or monomer-dependent interactions of a candidate molecule with the aperture are provided.
In another aspect of the invention, a method for evaluating a polymer molecule, the polymer molecule including linearly connected monomer residues includes providing a polymer molecule in a liquid, contacting the liquid with an insulating solid-state substrate having a detector capable of detecting polymer molecule characteristics, causing the polymer molecule to traverse a limited volume on the solid-state substrate so that monomers of the polymer molecule traverse the limit volume in sequential order, whereby the polymer molecule interacts linearly with the detector and data suitable to determine polymer molecule characteristics are obtained.
In some embodiments, the detector is an electrode, and electron current is detected as the monomer traverses the limited volume. The detector is a metal electrode located on the substrate surface, and further includes a polymer replicating catalyst attached to the solid-state surface adjacent to the detector, whereby the polymer replicating catalyst acts upon the polymer molecule, so that the polymer molecule interacts linearly with the detector as it advances through the polymer replicating catalyst. The polymer is selected from the group consisting of polynucleic acids, polynucleotides, DNA and RNA, and the liquid solution further includes reagents necessary to replicate the polymer molecule.
In one embodiment, the limited volume of the solid-state substrate is a groove on the solid-state substrate surface, and the detector is located at the base of the groove, whereby the polymer molecule traverses length of the groove.
In another aspect of the invention, a method for evaluating a polymer molecule including linearly connected monomer residues is provided. A candidate polymer molecule in a liquid is provided and contacted with an insulating solid-state membrane having an aperture therein, wherein the aperture includes an entry port and an exit port defining a channel there between. The candidate polymer molecule traverses the aperture of the membrane, whereby the polymer molecule interacts linearly with the aperture and data suitable to determine polymer molecule characteristics are obtained.
In some embodiments, polymer molecule interactions with the aperture are detected as electronic currents at first and second electrodes adjacent to the aperture and in electrical communication with said channel, or polymer molecule interactions with the aperture are detected by measuring ionic conductance in the channel. Translational current is detected, or current along the length of the channel is detected.
In some embodiments, the polymer molecule traverses the aperture by application of a voltage or use of a biomotor.
In some embodiments, the amplitude of duration of individual conductance measurements is indicative of sequential identity of monomers of the polymer molecule, or the number of changes in the conductance measurement is an indication of the number of monomers in the polymer, the duration of the individual conductance measurement is an indication of the number of monomers in the polymer molecule, or multiple molecules of a heterogeneous mixture of individual polymer molecules are characterized to provide a size distribution of polymers in the mixture.
In other embodiments, a polymer replicating catalyst is in contact with the aperture, and the polymer replicating catalyst is located adjacent to, below, above, or within.
In another aspect of the invention, a method for evaluating a polymer molecule including linearly connected monomer residues includes providing a candidate hybridized polynucleotide molecule in a liquid; and contacting the liquid with an insulating solid-state membrane having an aperture therein, said aperture having a diameter insufficient to permit traversal of the hybridized molecule of the aperture. The candidate polymer molecule traverses the aperture of the membrane, whereby the hybridized polymer molecule is denatured and the single-stranded polymer interacts linearly with the aperture and data suitable to determine polymer molecule characteristics are obtained.
In one embodiment, the hybridized polymer molecule oscillates between a first condition at which the polymer cannot advance into the aperture and a second condition at which the hybridized molecule is denatured and a single strand of the polymer advances into the aperture. The rate of oscillation between the first and second conditions is selected to advance the polymer by about a single monomer with each oscillation. The condition varied is an applied potential gradient across the membrane.
In other embodiments, the rate of traversal of a single strand DNA is an order of magnitude slower when using hybridized polymer than when using a single strand polymer.
The present invention provides a solid-state system for interacting with the polymeric molecule which overcome the limitations of the prior currently available biological pores, such as limited temperature and bias voltage operating ranges, limited chemical environment accommodation, limited device lifetimexe2x80x94due to pore diffusion in the membrane, high electronic noise levels associated with large membrane capacitance, and limited availability of pores with the desired diameter and lengths on the 1-10 nm scale.